p16^INK4a Methylation in Serum as a Follow-up Marker for Recurrence of Colorectal Cancer

Materials and Methods

Sample collection and DNA preparation. Peripheral blood samples were collected from 20 healthy donors and from 21 sporadic CRC patients who underwent surgical resection at Nagoya University Hospital, Nagoya, Japan. The CRC patient blood samples were obtained on the day before surgery and during the follow-up period at 2 weeks and 1, 3 and 6 months after surgical intervention. Tumor and corresponding normal tissue samples were obtained at the time of surgery from the 21 primary CRC and from two metastatic CRC. Written informed consent, as approved by the institutional review board, was obtained from all the patients. The tumor, normal tissue and serum samples were immediately frozen and stored at −80°C until the DNA was extracted. The samples were digested with proteinase K and the DNA was extracted.

Bisulfite modification. The DNA samples (1 μg) were denatured by NaOH and modified by sodium bisulfite as described elsewhere (10). The DNA samples were then purified using Wizard purification resin (Promega Corp, Madison, WI, USA), treated again with NaOH, precipitated with ethanol, then resuspended in water.

Quantitative methylation-specific PCR. Q-MSP was performed in an ABI sequence detection system 7000 using two amplification primers and a dual-labeled fluorogenic hybridization probe (Applied Biosystems, Foster City, CA, USA). The p16^INK4a primers for RTQ-PCR have been described elsewhere (11). The sense and antisense primers and fluorogenic probe for the methylated sequence were p16^INK4a MS (5’-TTATTAGAGGGTGGGGCGGATCGC-3’), p16^INK4a MAS (5’-GACCCCGAACCGCGACCGTAA-3’) and p16^INK4a MT [5’-(FAM)-AGTAGTATGGAGTCGGCGGCGGG-(TAMRA)-3’], respectively. The sense and antisense primers and fluorogenic probe for the unmethylated sequence were p16^INK4a US (5’-TTATTAGAGGGTGGGGTGGATTGT-3’), p16^INK4a UAS (5’-CAACCCCAAACCACAACCATAA-3’) and p16^INK4a UT [5’-(FAM)-AGGTAGTGGGTGGTGGGGAGTAGTATGGAGμTTG-(TAMRA)-3’], respectively.

Thermocycling was performed in a final volume of 50 μl containing 5.0 μl of bisulfite-converted DNA from 180 μl of serum sample, 900 nM each of the p16 primers (sense and antisense), 300 nM of the corresponding fluorogenic probe, 5.0 μl PCR buffer, 2.5 μl dNTP mix, and 1.5 units of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). The PCR amplification consisted of 50 cycles (95°C for 15 sec, 60°C for 60 sec, and 72°C for 18 sec) after an initial denaturation step (95°C for 10 min). Multiple negative water blanks were included in each analysis.

The amplification data were analyzed using the Sequence Detection System software developed by Applied Biosystems. The standard curve was established for quantifying the DNA copy number, using 9 known copy numbers of serially diluted (1 to 10⁸ copies) plasmids containing p16^INK4a methylated or unmethylated DNA. The Q-MSP assays were conducted in triplicate for each sample and the mean value (Q) was used for further calculation. The concentration (C) of p16 methylated or unmethylated DNA in the serum (genome-equivalents/μl) should be Q/180 because the 5.0 μl of bisulfite-converted DNA applied to RTQ-PCR was derived from 180 μl of serum. p16^INK4a methylation score (p16^INK4aMS) was defined as: p16^INK4a MS (%)=CM/(CM+CU) ×100, where CM=concentration of p16^INK4a methylated DNA in the serum and CU=concentration of p16^INK4a unmethylated DNA in the serum.